Methods for treating motor neuron disease

ABSTRACT

An object of the present invention is to provide an agent effective for the treatment and/or prevention of motor neuron disease such as amyotrophic lateral sclerosis (ALS). The present invention provides a therapeutic and/or preventive agent for motor neuron disease comprising the following oligopeptide shown in any of (a) to (c) or a pharmaceutically acceptable salt thereof as an active ingredient: (a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); (b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene; and (c) a modified oligopeptide from the oligopeptide (a) or (b).

This is a divisional application of copending U.S. application Ser. No. 11/578,141, which is the national phase of PCT International Application No. PCT/JP2004/018677 filed on Dec. 8, 2004, which claims priority under 35 U.S.C. §119(e) on U.S. Provisional Application No. 60/560,254 filed on Apr. 8, 2004; the entire contents of all are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a therapeutic and/or preventive agent for motor neuron disease, particularly amyotrophic lateral sclerosis (ALS).

BACKGROUND ART

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that typically affects people of middle or advanced ages and selectively attacks motor nerves in the cerebrum, brain stem, and spinal cord (Cleveland D W and Rothstein J D, 2001, Nat Rev Neurosci 2: 806-819; and Hand C K and Rouleau G A, 2002, Muscle Nerve 25: 135-159). ALS causes muscular atrophy and muscular weakness in voluntary muscles in the whole body except for extraocular muscle, and eventually respiratory failure. Most patients die in 3 to 5 years from the onset.

Riluzole is the sole drug previously approved for ALS in US and Japan. Riluzole was originally developed as an anticonvulsant inhibiting glutamate release and has been reported in several clinical trials to exhibit only slight efficacy for the survival of ALS patients (Rowland L P and Shneider N A, 2001, N Engl J Med, 344, 1688-1700; and Turner M R and Parton M J, 2001, Semin Neurol 21: 167-175). Besides riluzole, multiple factors including ciliary neurotrophic factor (CNTF) and insulin-like growth factor I (IGF-I) were tested in clinical trials and, however, fell short of success (Miller R G et al., 1996, Ann Neurol 39: 256-260). Thus, there are no therapeutic agents effective for ALS under present circumstances.

Approximately 10% of ALS cases are familial (FALS) and most FALS cases are inherited autosomal-dominantly. In 1993, Rosen et al identified for the first time the superoxide dismutase-1 (SOD1) gene located on the chromosome 21 as a causative gene by analyzing pedigree with autosomal inheritance (Rosen D R, et al., 1993, Nature 362: 59-62). Approximately 20% of FALS cases are caused by mutations in the SOD1 gene, and most of these mutations are missense point mutations. 100 or more mutations in SOD1 caused FALS (Cleveland D W and Rothstein L D, supra). Several groups have reported that overexpression of FALS-associated SOD1 mutant gene induces neuronal cell death in vitro (e.g. Rabizadeh S, et al., 1995, Proc Natl Acad Sci USA 92: 3024-3028; Durham H D et al., 1997, J Neropathol Exp Neurol 56: 523-530; and Ghadge G D et al., 1997 J Nerosci 17: 8756-8766). Besides this, the activation of caspase-3 has been observed in the spinal cords in ALS patients. Accordingly, the inhibition of neuronal cell death is an important strategy to develop therapeutic agents for ALS.

In addition to CNTF and IGF-I described above, Bcl2, a non-specific caspase inhibitor zVAD-fmk (Kostic V, et al., 1997, Science 277: 559-562; Azzouz M, et al., 2000; Hum Mol Genet. 9: 803-811; and Li M, et al., 2000, Science 288: 335-339), and alsin, the newly found product of ALS2 gene causative of recessive inherited FALS (Kanekura K, et al, 2004; J Biol Chem 279: 19247-19256), have been reported so far as those exhibiting inhibitory (antagonistic) action on neuronal cell death caused by the overexpression of SOD1 mutants.

On the other hand, ADNF or ADNF9 (activity-dependent neurotrophic factor); which is a short peptide consisting of nine amino acid residues (Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala), was originally purified by Gozes et al from the culture medium of astrocytes stimulated with VIP (Brenneman D E and Gozes I, 1996, J Clin Invest 97: 2299-2307; Brenneman D E, et al., 1998, J Pharmacol Exp Ther 285: 619-627; and Blondel O, et al., 2000, J Neurosci 20: 8012-8020). ADNF was shown to protect neurons from death caused by some neurological disorders including amyloid β (Brenneman D E, et al., 1998, J Pharmacol Exp Ther 285: 619-627; and Glazner G W, et al., 2000, J Neurochem 73: 2341-2347). ADNF is a neuroprotective factor unique in that it has activity at its lower concentrations of femtomolar to picomolar levels, and loses its protective effect at higher concentrations above the nanomolar order. This unique but unfavorable property of ADNF have prevented it from being developed as an anti-Alzheimer's disease (AD) drug.

The object of the present invention is to provide an agent that inhibits neuronal cell death causing ALS and is effective for the treatment of amyotrophic lateral sclerosis.

DISCLOSURE OF THE INVENTION

We have now conducted diligent studies to attaint the object and have consequently completed the present invention by finding out that an activity-dependent neurotrophic factor (hereinafter, referred to as “ADNF”) significantly inhibits (antagonizes) neuronal cell death induced by mutant superoxide dismutase-1 (SOD1) genes and has the effect of improving the motor function of ALS model mice and delaying ALS onset.

Namely, the present invention encompasses the following inventions.

(1) A therapeutic and/or preventive agent for motor neuron disease comprising the following oligopeptide shown in any of (a) to (c) or a pharmaceutically acceptable salt thereof as an active ingredient:

(a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1);

(b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene; and

(c) a modified oligopeptide from the oligopeptide (a) or (b).

(2) A therapeutic and/or preventive agent for motor neuron disease comprising DNA encoding the following oligopeptide (a) or (b) as an active ingredient:

(a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); and

(b) an oligopeptide consisting of an amino acid sequence having a a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene.

(3) The agent according to (1) or (2), wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS). (4) The agent according to (1) or (2), wherein the agent has the effect of delaying the onset of the motor neuron disease and improving the motor function of a patient with the motor neuron disease. (5) A fusion peptide of the following oligopeptide (a) or (b) with another peptide:

(a) an oligopeptide consisting, of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); and

(b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1) with the and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene.

(6) The fusion peptide according to (5), wherein the another peptide is a signal peptide for extracellular secretion and/or tag peptide for purification/detection. (7) DNA encoding the following oligopeptide (a) or (b) or a fusion peptide according to (5):

(a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or

(b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene.

(8) A recombinant vector comprising DNA according to (7). (9) A host cell transformed with DNA according to (8). (10) A method for producing the following oligopeptide (a) or (b), characterized by culturing a host cell according to (9) in a medium and collecting an oligopeptide expressed from the obtained culture:

(a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1); or

(b) an oligopeptide consisting of an amino acid sequence having a deletion, substitution, insertion, or addition of one or several amino acids in Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1), and having an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 gene.

The present invention also encompasses a use of the oligopeptide shown in any of (a) to (c) or a pharmaceutically acceptable salt thereof for the production of said agent; and a method for treating a motor neuron disease comprising the step of administering an effective amount of the oligopeptide shown in any of (a) to (c) or a pharmaceutically acceptable salt thereof to a mammal including human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows the cell mortality of a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS vector) or wild-type SOD1 gene (wt-SOD1) FIG. 1(B) shows the cell mortality of a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or SOD1 mutant (G85R-SOD1) gene. FIG. 1(C) shows the cell mortality of a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS), wild-type SOD1 gene (wt-SOD1), or SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene, in the presence or absence of ADNF or HNG;

FIG. 2A shows the dose-dependent inhibitory effect of ADNF on cell death induced by a neuronal cell line (F11 cell) transformed with a SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 2B shows the dose-dependent inhibitory effect of ADNF on cell death induced by a neuronal cell line (NSC34 cell) transformed with a SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 3A shows the effect of wortmannin (W), genistein (G), PD98059 (PD), SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (A4T-SOD1) gene, in the presence or absence of 100 nM ADNF. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 3B shows the effect of wortmannin (W), genistein (G), PD98059 (PD), SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (G85R-SOD1) gene, in the presence or absence of 100 nM ADNF. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 3C shows the effect of wortmannin (W), genistein (G), PD98059 (PD), SB203580 (SB), AG490 (AG), KN93 (KN), or HA1004 (HA) on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (G93R-SOD1) gene, in the presence or absence of 100 nM ADNF. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 4A shows the effect of KN93 or KN92 on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (A4T-SOD1) gene, in the presence or absence of 100 fM ADNF. The lower panel shows the expression level of SOD1 protein detected by immunoblot;

FIG. 4B shows the effect of KN93 or KN92 on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (G85R-SOD1) gene in the presence or absence of 100 fM ADNF. The lower panel shows the expression level of SOD1 protein detected by immunoblot;

FIG. 4C shows the effect of KN93 or KN92 on cell death induced by a neuronal cell line (F11 cells) transfected with a control vector (pEF-BOS) or with a SOD1 mutant (G93R-SOD1) gene in the presence or absence of 100 fM ADNF. The lower panel shows the expression level of SOD1 proteins detected by immunoblot;

FIG. 4D shows the cell death of F11 cells, which were cotransformed with a SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene and with a kinase-inactive CaMKII or CaMKIV cDNA, in the presence or absence of 100 nM ADNF;

FIG. 5A shows the effect of an IPAL peptide (IPALDSLKPANEDQKIGIEI) on cell death induced by a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS vector) or with a SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) gene in the presence or absence of 100 fM ADNF;

FIG. 5B shows the cell mortality of a neuronal cell line (F11 cell) transformed with a control vector (pEF-BOS) or with a SOD1 mutant (G93R-SOD1) gene in the presence of varying concentrations of ADNF (SALLRSIPA) or ADNF8 (ALLRSIPA); and

FIG. 6 shows the effect of icy injection of ADNF on the motor function of G93A-SOD1 transgenic mice.

Hereinafter, the present invention will be described in detail. The present application claims the priority of U.S. Provisional Application No. 60/560,254 filed on Apr. 8, 2004 and encompasses contents as described in the specification and/or drawings of the priority application.

1. Agent of the Invention

An oligopeptide, as an active ingredient, in the agent of the present invention is an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 1).

The oligopeptide also encompasses a mutant oligopeptide having a deletion, substitution, insertion, or addition of one or several amino acids in said amino acid sequence as long as the mutant oligopeptide has an activity that inhibits neuronal cell death caused by a mutant superoxide dismutase-1 (hereinafter, referred to as “SOD1 mutant) gene. The range of the “one or several” is not particularly limited and means, for example, one to five, preferably one to three, more preferably one or two amino acids.

The substation by another amino acid can include the substitution between hydrophobic amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Thr, and Val), between hydrophilic amino acids (Arg, Asp, Asn, Cys, Glu, Gln, Gly, His, Lys, Ser, and Thr), between amino acids with aliphatic side chains (Gly, Ala, Val, Leu, Ile, and Pro), or between amino acids with hydroxy group-containing side chains (Ser, Thr, and Tyr). However, these substitutions are merely illustrated for preferable examples, and other amino acid substitutions may be performed as long as the resulting mutant oligopeptide maintains an activity that inhibits neuronal cell death caused by a SOD1 mutant gene.

The SOD1 mutant gene refers to a gene encoding SOD1 comprising a substitution of Ala at position 4 (corresponding to position 5 in SEQ ID NO: 5) by Thr, a substitution of Gly at position 85 (corresponding to position 86 in SEQ ID NO: 5) by Arg, or a substitution of Gly at position 93 (corresponding to position 94 in SEQ ID NO: 5) by Arg, provided that the position of Ala following the initiation codon (Met) is numbered 1, in the wild-type human SOD1 amino acid sequence (SEQ ID NO: 5).

The oligopeptide and an altered oligopeptide thereof may be modified chemically or biologically. Examples of the modification can include, but are not limited to, functional group introduction such as alkylation, esterification, halogenation, or amination, functional group conversion such as oxidation, reduction, addition, or elimination, the introduction of sugar compounds (monosaccharide, disaccharide, oligosaccharide, or polysaccharide) or lipid compounds, phosphorylation, and biotinylation.

Those skilled in that art can confirm by test methods as described specifically and in detail in Examples later or by appropriately altered or modified versions of the test methods that the altered oligopeptide or modified oligopeptide has an activity that inhibits cell death caused by a SOD1 mutant gene, which activity is analogous to the inhibitory activity of the oligopeptide comprising the amino acid sequence represented by SEQ ID NO: 1.

The oligopeptides are of various types (including altered or modified oligopeptides), and they may be in a free form or in an acid- or base-addition salt. Examples of the acid-addition salt can include salts of mineral acids such as hydrochloride, sulfate, nitrate, and phosphate; and salts of organic acids such as citrate, oxalate, maleate, and tartrate. Examples of the base-addition salt can include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts; ammonium salts; organic ammonium salts such as methylammonium salts and triethylammonium salts.

Furthermore, these oligopeptides or salts thereof sometimes exist as hydrates or solvates.

The oligopeptides can be synthesized by a routine peptide synthesis method known in the art. Specifically, they can be synthesized by a variety of methods such as azide method, acid chloride method, acid anhydride method, mixed anhydride method, DCC method, activated ester method (e.g., P-nitrophenyl ester, N-hydroxysuccinimide ester, and cyanomethyl ester methods), methods using Woodward's reagent K, carboimidazole method, oxidation-reduction method, and DCC-additive (HONB, HOBt, or HOSu) method according to the descriptions of, for example, “The Peptides” Vol. 1 (1966) [Schroder and Lubke, Academic Press, New York, U.S.A.] or “Peptide Synthesis” [Izumiya et al., Maruzen Co., Ltd., (1975)]. These methods can be applied to both solid-phase and liquid-phase syntheses.

In the solid-phase method, a variety of commercially available peptide synthesizers can be utilized. The synthesis can be performed more efficiently by protecting and deprotecting functional groups, if necessary. For example, Protective Groups in Organic Synthesis (T. W. Greene, John Wiley & Sons Inc., 1981) can be referenced for procedures for introducing and eliminating protecting groups.

The obtained oligopeptide can be desalted and purified according to a typical method. Examples thereof include ion-exchange chromatography such as DEAE-cellulose, partition chromatography such as Sephadex LH-20 and Sephadex G-25, normal phase chromatography such as silica gel, reverse phase chromatography such as ODS-silica gel, and high performance liquid chromatography.

The oligopeptide inhibits neuronal cell death caused by a SOD1 mutant gene and has neuroprotective action. The neuroprotective action of the oligopeptide is attributed to a mechanism (novel mechanism) different from that of antagonistic action on neuronal cell death caused by previously reported amyloid βtoxicity because the action was not inhibited by IPAL peptides (Example 5 below) and was exhibited even after the deletion of one N-terminal amino acid of the oligopeptide (Example 6 below).

Thus, when the oligopeptide is used for disease that collapses the motor neuron mechanism due to neuronal cell death caused by a SOD1 mutant gene, for example amyotrophic lateral sclerosis (ALS), the oligopeptide can remedy the disease by inhibiting the neuronal cell death. As used herein, the “motor neuron disease” refers to a neurodegenerative disease with progressive, retrograde disorder of upper and lower motor neurons that control motion in the body. Examples of the disease typically include amyotrophic lateral sclerosis (ALS) and also include, but not limited to, spinal muscular atrophy (SMA: Werdnig-Hoffmann disease or Kugelberg-Welander syndrome) and bulbospinal muscular atrophy (BSMA: Kennedy-Alter-Sung syndrome). The agent of the present invention has efficacy as a preventive agent preventing or delaying the onset of the motor neuron disease and/or a therapeutic agent allowing the motor neuron disease to recover to the normal state. The agent of the present invention is also effective for the amelioration (or improvement) of conditions resulting from the motor neuron disease. The amelioration of conditions refers to the amelioration of, for example, muscular atrophy, muscular weakness, bulbar palsy (muscular atrophy or weakness in the face, pharynx, and tongue, and aphasia or dysphagia caused thereby), muscular fasciculation, and respiratory disorder.

The agent of the present invention can be provided as a pharmaceutical composition by preparing a purified preparation of the oligopeptide into various types of dosage forms by a variety of methods known in the art. The agent of the present invention, when orally administered, may be prepared into tablets, capsule, granules, powders, pills, liquors for internal use, suspensions, emulsions, syrups, or the like, or may be made into dry products which are redissolved when used. Alternatively, the agent of the present invention, when parenterally administered, is prepared into intravenous injections (including infusion), intramuscular injections, intraperitoneal injections, hypodermic injections, suppositories, or the like, and the agent used as a preparation for injection is provided in the form of unit dose ampule or multiple dose container.

Various types of these preparations can be produced by a routine method by appropriately selecting excipients, fillers, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavors, soothing agents, stabilizers, tonicity agents, and so on, typically used for preparations. The content of the oligopeptide as an active ingredient in the pharmaceutical composition may be on the order of, for example, 0.1 to 10% by weight.

The agent of the present invention, when used as a preventive and/or therapeutic agent for the disease described above, can be administered parenterally or orally with safety to mammals such as humans, mice, rats, rabbits, dogs, and cats. The dose of the agent of the present invention may be changed appropriately depending on the ages of individuals to be administered, administration routes, and the number of doses. For example, the effective amount of the oligopeptide combined with suitable diluents and pharmacologically available carriers is, for example, in the range of 1 to 500 μg/kg body weight/day.

The active ingredient of the agent of the present invention may be DNA (or a gene) encoding the oligopeptide. When the gene encoding the oligopeptide is used as a gene therapy agent for the disease described above, examples of administration methods thereof include a method which directly administers the gene by injection and a method which administers a vector incorporating the gene therein. Examples of the vector include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, and retrovirus vectors. Efficient administration can be achieved by using these virus vectors. Alternatively, a method which introduces the gene into phospholipid vesicles such as liposomes and administers the liposome may be used.

The administration mode of the gene-therapeutic agent may be any of local administration such as administration to quadriceps femoris muscle or gluteus maximus and systemic administration such as typical intravenous or intraarterial administration and is preferably local administration. Furthermore, the administration mode combined with catheter techniques, surgical operation, and so on, can be adopted.

2. Expression System of DNA Encoding Oligopeptide

The oligopeptide used in the agent of the present invention can also be produced according to typical genetic engineering techniques. Namely, a recombinant vector comprising the DNA encoding the oligopeptide is constructed, and a microorganism (transformant) transformed with the vector is prepared. The desired oligopeptide can be separated and purified from a culture obtained by culturing the transformant.

In the oligopeptide production by the genetic engineering techniques, the oligopeptide can be secreted actively outside of the host cell by expressing it in the form of a fusion peptide of the oligopeptide with a signal peptide for extracellular secretion added to the N terminus thereof. Furthermore, a tag for purification/detection can be added to between the signal peptide and the oligopeptide or to the C-terminus of the oligopeptide.

The production of the fusion protein may be performed by procedures in which the DNA encoding the oligopeptide and DNA encoding another peptide are ligated in frame and introduced into an expression vector to express the fusion protein in a host. Approaches already known in the art can be used.

Any signal peptide of secretion proteins known in the art, which is selected depending on the types of host cells, can be used as the signal peptide of the present invention. When animal cells are used as host cells, examples of the signal peptide include signal peptides present in the N termini of growth and differentiation factors (e.g., a variety of cytokines) and receptors thereof.

Any of those known in the art can be used as the tag for purification/detection, and examples thereof include FLAG, 6×His, 10×His, influenza hemagglutinin (HA), VSV-GP fragments, T7-tag, HSV-tag, and E-tag.

Vectors containing DNAs encoding these peptide sequences as inserts and each host (Escherichia coli, yeast, and animal cell) are commercially available.

The recombinant vector of the present invention can be obtained by ligating the gene encoding the oligopeptide to an appropriate vector. The vector into which the gene is inserted is not particularly limited as long as it enables replication in hosts. Examples of the vector include plasmid DNA and phage DNA. Examples of the plasmid DNA include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, and yeast-derived plasmids. Examples of the phage DNA include phages. Furthermore, animal viruses such as retrovirus or vaccinia virus and insect virus vectors such as baculovirus can also be used.

To insert the gene into a vector, a method which initially cleaves the purified DNA with appropriate restriction enzymes and inserts and ligates it at the restriction site or multicloning site of appropriate vector DNA can be adopted.

The DNA encoding the oligopeptide is incorporated into the vector so that the function of the DNA is exerted. Thus, the vector of the present invention can be ligated, if desired, with those containing cis elements such as enhancers, splicing signals, polyA-addition signals, selective markers, ribosome-binding sequences (SD sequences), and so on, in addition to promoters and the DNA. Examples of the selective markers include dihydrofolate reductase genes, ampicillin resistance genes, and neomycin resistance genes.

The host cell (i.e., transformant) of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the DNA of interest can be expressed. In this context, the host is not particularly limited as long as it can express the gene. Examples of the host include bacteria belonging to the genus Escherichia such as Escherichia coli and the genus Bacillus such as Bacillus subtilis; yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; and animal cells such as monkey COS-7 cell, Vero, Chinese hamster ovary cell (CHO cell), mouse L cell, human GH3 cell, and human FL cell.

Examples of methods for introducing the recombinant vector into the host include electroporation, calcium phosphate, lithium acetate, lipofection, and virus methods, which are selected depending on the types of hosts. Methods independent of recombinant vectors, for example particle gun method, can also be used for gene delivery to each of the host cells.

The oligopeptide can be obtained by culturing the transformant, followed by collection from the resulting culture. The “culture” means any of culture supernatants, cultured cells or cultured microorganisms, and homogenates of the cultured cells or cultured microorganisms.

The transformant of the present invention can be cultured in a medium according to a method typically used for culturing the host cell.

Both natural and synthetic media may be used as the medium for culturing the transformant obtained from a microorganism such as Escherichia coli or yeast used as a host as long as the media contain carbon sources, nitrogen sources, inorganic salts, and so on capable of being assimilated by the microorganism and can achieve the efficient culture of the transformant. The carbon sources may be those capable of being assimilated by the microorganism. Carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. Ammonium salts of inorganic or organic acids (e.g., ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate), other nitrogen-containing compounds, peptone, meat extracts, corn steep liquor, and so on can be used as the nitrogen sources. Monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and so on can be used as the inorganic salts.

When the oligopeptide is produced into the cultured microorganism or cells, the oligopeptide is extracted by homogenizing the microorganism or cells. When the oligopeptide is produced outside of the microorganism or cells, the culture liquid is directly used or, for example, centrifuged to remove the microorganism or cells. Then, the oligopeptide of interest can be isolated and purified from the culture by using, alone or in appropriate combination, general biochemical methods used in protein isolation and purification, for example ammonium sulfate precipitation, gel chromatography, ion-exchange chromatography, and affinity chromatography.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more fully with reference to Examples. However, the present invention is not intended to be limited by these Examples.

All of experiments using neuronal cells lines described in Examples below were repeated at least three times with independent transformations and treatments, each of which gave essentially the same results. Statistical analysis was conducted by one-way ANOVA, followed by Bonferroni/Dunn post-hoc test, in which p<0.05 was assessed as being significant.

Animal experiments were conducted according to Policies on the Use of Animals and Humans in Neuroscience Research, the Society for Neuroscience and Guideline for Care and Use of Laboratory Animals of KEIO University. All experimental procedures were approved by Institutional Animal Experiment Committee at KEIO University.

Example 1 Inhibition of SOD1 Mutant Gene-Induced Neuronal Cell Death by ADNF (1) Test Materials

Wild-type SOD1 cDNA (SEQ ID NO: 4) and SOD1 mutant (A4T-SOD1, G85R-SOD1, and G93R-SOD1) cDNAs were kindly provided by Dr. Shoji Tsuji (Faculty of Medicine, the University of Tokyo). Kinase-inactive CaMKII and CaMKIV cDNAs were kindly provided by Dr. Howard Schulman, Stanford University, US. ADNF (SALLRSIPA: SEQ ID NO: 1) and ADNF8 (ALLRSIPA: SEQ ID NO: 2) were synthesized (Glazner G W, et al., 1999, J Neurochem 73: 2341-2347). IPAL peptide (IPALDSLKPANEDQKIGIEI: SEQ ID NO: 3, Zamostiano R, et al., 1999, Neurosci Lett 264: 9-12) was purchased from the Peptide Institute (Osaka, Japan). An anti-SOD1 antibody was purchased from MBL (Nagoya, Japan). PD98059, SB20380, AG490, KN93, KN92, and HA1004 were purchased from Calbiochem-Novabiochem (San Diego, USA).

F11 cell, the hybrid cell of rat embryonic day 13 (E13) primary cultured neuronal cell with mouse neuroblastoma NTG18 cell, was cultured in Ham's F-12 medium (Life Technologies, Gaithersburg, Md.) containing 18% FBS (Hyclone, Logan, Utah) and antibiotics as previously reported (Platika D, et al., 1985, Proc Natl Acad Sci USA 82: 3499-3503; Yamatsuji T, et al., 1996, Science 272: 1349-1352; Huang P, et al., 2000, Mol Hum Reprod 6: 1069-1078; and Niikura T, et al., 2001, J Neuroscience, 21: 1902-1910).

NSC34 cell, the hybrid cell of primary cultured, motor neuron-system embryonic mouse spinal cord cell with mouse neuroblastoma NTG18 cell, was cultured in DENTE medium containing 10% FBS and antibiotics (Cashman N R, et al., 1992, Dev Dyn 194: 209-221; and Durham H D, et al, 1993, Neurotoxicology 14: 387-395).

(2) Test Methods (2-1) Neuronal Cell Death Test

The F11 cells (7×10⁴ cells/well, 6-well plate, 12- to 16-hour culture in Ham's F-12 (18% PBS) medium) were transformed with wild-type or SOD1 mutant (A4T, G85R, and G93R) genes by lipofection (0.5 μg of each SOD1 gene; 1 μl of LipofectAMINE; 2 μl of PLUS Reagent) under serum-free conditions for 3 hours and cultured for 2 hours in Ham's F-12 (18% PBS) medium. The medium was replaced by Ham's F-12 (10% PBS) in the presence or absence of ADNF. After 72 hours of the transformation, cell mortality was measured by Trypan blue exclusion assay as previously reported (Hashimoto Y, et al., 2001, J Neurosci 21: 9235-9245; and Hashimoto Y, et al, 2001, Proc Natl Acad Sci USA 98: 6336-6341).

The NSC34 cells (7×10⁴ cells/well, 6-well plate, 12 to 16-hour culture in DMEM (10% FBS) medium) were transformed with wild-type or SOD1 mutant (A4T, G85R, and G93R) genes by lipofection (0.5 μg of each SOD1 gene; 1 μl of LipofectAMINE; 2 μl of PLUS Reagent) under serum-free conditions for 3 hours and cultured for 21 hours in DMEM (10% FBS) and after 48 hours in DMED supplemented with N2 supplement (Invitrogen). After 72 hours of the transformation, cell mortality was measured by Trypan blue exclusion assay in the same way as above.

(2-2) Immunoblot Analysis

Immunoblot analysis was conducted according to the previous report (Hashimoto Y, et al., supra, p. 6336-6341). To examine the protein expression of wild-type or SOD1 mutant genes, lysates from the cells transfected with each SOD1 gene were subjected to SDS-PAGE (20 μg/lane). After electrical blotting to a PVDF membrane, the membrane was blocked by a typical method and reacted with an anti-SOD1 antibody and then with a 1:5000-diluted horseradish peroxidase-conjugated anti-mouse IgG antibody (Bio-Rad Lab. Hercules, Calif., USA). The antibody-reactive bands were detected by ECL (Amersham Pharmacia Biotech, Uppsala, Sweden).

(3) Result (3-1) Confirming Induction of Neuronal Cell Death of F11 Cells by SOD1 Mutant Gene Transfer

F11 cells were transformed with 0.25, 0.5, or 1.0 μg of wild-type SOD1 (wt SOD1) cDNA or SOD1 mutant (G85R-SOD1) cDNA. After 72 hours, cell mortality was measured by Trypan blue exclusion assay. The pEF-BOS vector was used as a control. Cell mortality induced by the transformation with 0.25 or 0.5 μg of wild type SOD1 cDNA was approximately 10%, which was similar to that induced by the transformation with the control vector. However, cell mortality induced by the transformation with 1 μg of the wild type SOD1 cDNA was 45%, indicating that even wild-type SOD1 causes cell death by its overexpression (FIG. 1(A)). On the other hand, cell mortality induced by the transformation with 0.25 μg of G85R-SOD1 cDNA was 17%, which was slightly higher than that induced by the transformation with the control vector, while cell mortalities induced by the transformation with 0.5 and 1.0 μg of G85R-SOD1 cDNA were 50% and 60%, respectively (FIG. 1(B)). Based on these results, the amount of cDNA for the transformation of three types of SOD1 mutant genes to induce the cell death of F11 cells was decided to be 0.5 μg.

(3-2) Effect of ADNF on Neuronal Cell Death Induced by SOD1 Mutant Gene

The effect of ADNF on neuronal cell death induced by SOD1 mutant genes was tested. For comparison, S14G Humanin (HNG) was used. S14G-HN (HNG) has been reported to prevent neuronal cell death caused by some Alzheimer's disease-associated disorders but fail to prevent neuronal cell death induced by SOD1 mutant genes (Hashimoto Y, et al., supra, p. 6336-6341).

F11 cells were transformed with wild-type or SOD1 mutant (A4T-SOD1, G85R-SOD1, and G93R-SOD1) genes (0.5 μg each) in the presence or absence of 100 fM ADNF or 10 nM HNG. After 72 hours, cell mortality was measured by Trypan blue exclusion assay. The pEF-BOS vector was used as a control.

The transformation with the SOD1 mutant genes resulted in the death of 40 to 50% of the cells (FIG. 1(C)). On the other hand, the transformation with the control vector caused the death of only 10% of the cells. The addition of 100 fM ADNF decreased the cell mortalities induced by these SOD1 mutant genes to the level of the control. By contrast, the addition of 10 nM HNG could not reduce the cell death induced by the SOD1 mutant genes (FIG. 1(C)).

Example 2 Dose-Dependent Inhibitory Effect of ADNF on Neuronal Cell Death

F11 or NSC34 cells were used to confirm the dose-dependent effect of ADNF on neuronal cell death induced by transformation with SOD1 mutant genes (A4T-SOD1, G85R-SOD1, and G93R-SOD1).

F11 or NSC34 cells were transformed with pEF-BOS, A4T-SOD1, G85R-SOD1, or G93R-SOD1 cDNA in the presence of increasing concentrations (10 aM, 1 fM, 100 fM, 10 pM, 1 nM, and 100 nM) of ADNF. After 72 hours, cell mortality was measured by Trypan blue exclusion assay.

Although 10 aM ADNF hardly exhibited cell death inhibition, 100 fM ADNF completely decreased cell mortalities induced by these three types of SOD1 mutant genes to the level of the control (FIG. 2A). In this regard, the complete protective action of ADNF on cell death caused by the SOD1 mutant genes was observed at the concentrations equal to or above 10 nM. These results demonstrated the dose-dependent inhibitory activity of ADNF against neuronal cell death caused by SOD1 mutant genes.

Similarly, the experiment using NSC34 cells also showed that 100 fM ADNF can completely inhibit neuronal cell death caused by the three types of SOD1 mutant genes (FIG. 2B). ADNF exhibited dose-dependent inhibitory activity against the neuronal cell death of NSC34 cells, as with the F11 cells, and its effect was not decreased even at the concentrations equal to or above 10 nM.

Furthermore, the tests using both cells also demonstrated that SOD1 gene expression levels are not affected by ADNF treatment.

ADNF has been reported to lose its neuroprotective action at or above 10 nM (Brenneman D E, et al., 1996, J Clin Invest 97: 2299-2307; and Brenneman D E, et al., 1998, J Pharmacol Exp Ther 285: 619-627). In agreement with this result, we have also confirmed that ADNF at or above nM levels possesses reduced inhibitory activity against cell death including neuronal cell death caused by amyloid β toxicity- or APP mutant gene-induced Alzheimer's disease-associated disorders (Hashimoto Y, et al., 2001, Proc Natl Acad Sci USA 98: 6336-6341).

Thus, the dose-dependent neuroprotective effect of ADNF confirmed in the present tests on neuronal cell death caused by SOD1 mutant was probably different from that on amyloid β neurotoxicity.

Example 3 Analysis on Neuroprotective Action of ADNF

Intracellular signaling by ADNF was examined. F11 cells were transformed with pEF-BOS or SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) cDNA and reacted with 10 nM wortmannin (PI3 kinase inhibitor), 100 μM genistein (tyrosine kinase inhibitor), 50 μM PD98059 (MEK inhibitor), 20 μM SB203580 (p38 MAPK inhibitor), 1 μM AG490 (JAK kinase inhibitor), 5 μM KN93 (calcium/calmodulin-dependent kinase inhibitor), or 10 μM HA1004 (protein kinase A inhibitor) in the presence or absence of 100 nM ADNF. After 72 hours of the transformation, cell mortality was measured by Trypan blue exclusion assay.

The results obtained using A4T-SOD1, G85R-SOD1, and G93R-SOD1 are respectively shown in FIGS. 3A to 3C.

The neuroprotective effect of ADNF was not affected by wortmannin (W), PD98059 (PD), SB203580 (SB), AG490 (AG), and HA1004 (HA), but it was inhibited by genistein (G) and KN93 (KN) (FIGS. 3A to 3C). This suggests that ADNF exerts its neuroprotective action via certain tyrosine kinase and calcium/calmodulin-dependent kinase (CaMK) but does not activate PI3 kinase, MEK, p38 MAPK, JAK2, and PKA for exhibiting the neuroprotective action.

Example 4 Involvement of CaMKIV in Downstream of Signaling Pathway of ADNF

The effect of KN93 on the neuroprotective action of ADNF was examined. F11 cells were transformed with pEF-BOS or SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) cDNA and reacted with KN93 (10 nM, 50 nM, 100 nM, 500 nM, 1 μM, or 5 μM) or 5 μM KN92, an inactive form of KN93, in the presence or absence of 100 fM ADNF. After 72 hours of the transformation, cell mortality was measured by Trypan blue exclusion assay.

The results obtained using, A4T-SOD1, G85R-SOD1, and G93R-SOD1 are respectively shown in FIGS. 4A to 4C.

The neuroprotective action of ADNF was partially inhibited by 50 nM KN93 and almost completely inhibited by 1 μM KN93. KN93 at any of the concentrations had no influence on cell mortality at the control level. As expected, inactive KN92 even at the concentration of 5 μM did not influence ADNF activity. The protein expression of SOD1 mutant detected by immunoblot was not affected by KN93 or KN92. This dose-dependent inhibition of KN93 was also observed in all of the cells transformed with A4T-SOD1, G85R-SOD1, and G85R-SOD1 (FIGS. 4A to 4C).

Next, the influence of a kinase-inactive form of CaMKII or CaMKIV on the neuroprotective action of ADNF was examined.

NSC34 cells were cotransformed with pSR-alpha, A4T-SOD1, G85R-SOD1 or G93R-SOD1 cDNA and with pSR-alpha, a kinase-inactive CaMKII cDNA, or a kinase-inactive CaMKIV cDNA, and further cultured in the presence or absence of 100 nM ADNF. After 72 hours from the transformation, the cell mortality was measured.

Neither of the control vector or the kinase-inactive CaMKII influenced the neuroprotective action of ADNF. By contrast, the kinase-inactive CaMKIV inhibited the neuroprotective action of ADNF (FIG. 4D). This suggests that CaMKIV, but not CaMKII, is located downstream of the signaling pathway of ADNF. Neither the kinase-inactive CaMKII nor the kinase-inactive CaMKIV influenced the cell mortality at the control level.

Example 5 Difference Between Neuroprotective Effect of ADNF on FALS-Associated Disorders and Protective Effect on Amyloid β Toxicity

The IPAL peptide (IPALDSLKPANEDQKIGIEI: SEQ ID NO: 3) has been reported to inhibit the protective effect of ADNF on the cell death induced by amyloid β (Zamostiano R, et al., 1999, Neurosci Lett 264: 9-12). Thus, the effect of the IPAL peptide on the neuroprotection of ADNF against cell death caused by SOD1 mutant genes was examined.

F11 cells were transformed with pEF-BOS or SOD1 mutant (A4T-SOD1, G85R-SOD1, or G93R-SOD1) cDNA and reacted with 100 fM ADNF in the presence of 10 μM IPAL peptide. After 72 hours of the transformation, cell mortality was measured. The IPAL peptide did not inhibit the inhibitory activity of ADNF against cell death caused by the three types of SOD1 mutant genes, and it did not influence the cell death caused by the SOD1 mutant genes (FIG. 5A). In this regard, the IPAL peptide itself did not influence the cell death at the control level.

Example 6 Effect of N-Terminally Truncated ADNF

ADNF8 (ALLRSIPA: SEQ ID NO: 2), which lost one N-terminal amino acid residue in ADNF, has been reported to be totally ineffective against neuronal cell death caused by amyloid β or TTX (Brenneman D E, et al., 1998 J Pharmacol Exp Ther 285: 619-627). Thus, the effect of ADNF8 on cell death caused by G93A-SOD1 was examined.

F11 cells were transformed with pEF-BOS or G93A-SOD1 cDNA and reacted with varying concentrations of ADNF (S ALLRSIPA) or ADNF8 (ALLRSIPA). After 72 hours of the transformation, cell mortality was measured. ADNF8 at the concentration of 10 pM completely inhibited cell death caused by G93R-SOD1, indicating that ADNF8 is effective, though 100-fold less than ADNF, for the inhibition of neuronal cell death caused by SOD1 mutant genes (FIG. 5B).

Example 7 Motor Performance Test with ALS Model Animal

(1) Animal used

Transgenic (Tg) mice (hereinafter, referred to as “G93A-SOD1 Tg mice”) expressing human FALS-associated SOD1 mutant gene with a mutation (G93A) from Gly to Ala at 93 position are the best-established mouse model of ALS (Gurney M E, et al., 1994, Science 264: 1772-1775; and Gurney M E, et al., 1997, J Neurol Sci 152 Suppl 1: S67-73). The G93A-SOD1 Tg mice manifest symptoms quite similar to human ALS after normal birth and rapidly result in death in all cases. The G93A-SOD1 Tg mice, which have the onset of the regression of motor neurons similar clinically and pathologically to that in human ALS, have been known so far to be the most excellent model of ALS throughout the world and employed for identifying effective candidate agents for ALS patients.

(2) Test methods

The model mice were used to confirm the in vivo effect of ADNF on neurotoxicity induced by G93A-SOD1.

The G93A-SOD 1 Tg mice were purchased from Jackson Laboratories (Bar Harbor, Me.). The G93A-SOD1 Tg mice were kept as hemizygote mice by the mating thereof with C57BL/6J mice (CLEA Japan, Inc). The mice were raised in a SPF room (specific pathogen-free animal facility; 23±1° C., 55±5% humidity) in the 12-hour light/½-hour dark cycle (7:00 AM-7:00 PM). The mice were freely fed with gamma ray-irradiated Picolab Rodent Diet 20 (PMI Feeds Inc. St. Louis, Mo.) and sodium hyposulfite (5 ppm)-containing aseptic deionized distilled water.

The G93A-SOD1 Tg mice at 10 weeks of age were put under anesthesia by the intraperitoneal injection of 10% sodium pentobarbital (60 mg/kg). A hole was made on the cranial bone with a drill on the operating table of a stereotactic instrument to aseptically transplant the C315GS-4 cannula system for mouse (Plastics One Inc., Roanoke, Va.) to the mouse left cerebral ventricle. The cannula was fixed with a surgical adhesive and dental cement. The mice at 80 days of age were divided at random to a saline (control)-administered group (n=8) and an ADNF-administered group (n=11), and 3 μl of saline and 3 μl of 30 nmol ADNF were intracerebroventricularly (icv) injected each day to the former and the latter groups, respectively, until the end of the experiment. The injection was performed with the cannula in C3151S-4 connected to Hamilton syringe through a cannula tube (C232, PE50/Thin wall, Plastic One).

The motor function (motor performance) was evaluated weekly with a rotarod (CLEA Japan Inc). After the cannulation, the mice were acclimated to the rotarod for 2 days. The mice were placed onto a rotating rod moving at 5 rpm, and the time for which each mouse could remain on the rod was automatically detected. The test was conducted according to the protocol wherein the test was completed as a score of 7 minutes if the mouse remained on the rod for 7 minutes (Li M, et al., 2000, Science 288: 335-339; and Kaspar B K, et al., 2003, Science 301: 839-842). Disease onset was defined as the first day when the mouse could not remain on the rotarod for 7 minutes.

Death was defined when the mouse was unable to right itself within 30 seconds after being placed on its back (Li M, et al., supra).

(3) Result

The ADNF-treated ALS mice had motor performance significantly better than that of the control mice on Week 16, suggesting that the ADNF-treated mice did not cause reduction in motor function and maintained their motor functions (FIG. 6). However, this difference between the control mice and the ADNF-treated mice disappeared on Week 18 to Week 20.

The disease onset and survival days of the saline- and ADNF-administered groups are shown in Table 1 below.

TABLE 1 Saline-administered ADNF-administered group (n = 8) group (n = 11) Disease onset days 122.5 ± 7.2 127.3 ± 2.6 Survival days 156.8 ± 3.5 155.3 ± 2.2

Disease onset in the ADNF-administered group was prone to be delayed, although no significant difference in disease onset was obtained between the saline-administered group and the ADNF-administered group. The mean survival days of the ALS control mice and the ADNF-treated group were 156.8±3.5 days and 155.3±3.7 days, respectively, showing no difference in survival rate between them.

All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides an agent that significantly inhibits (or antagonizes) neuronal cell death induced by superoxide dismutase-1 (SOD1) mutant genes and has the effect of delaying the onset of motor neuron diseases including amyotrophic lateral sclerosis (ALS) and improving the motor function of a patient with motor neuron disease. Thus, the agent of the present invention is useful for the treatment and/or prevention of motor neuron diseases. 

1. A method for treating or preventing motor neuron disease comprising: administering to a mammal an effective amount of (a) an oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO. 1); (b) an oligopeptide consisting of an amino acid sequence represented by Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 2); or (c) a pharmaceutically acceptable salt thereof.
 2. The method according to claim 1, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS).
 3. The method according to claim 1, wherein (a) the oligopeptide consisting of the amino acid sequence represented by Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO. 1); (b) the oligopeptide consisting of an amino acid sequence represented by Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO: 2); or (c) the pharmaceutically acceptable salt thereof is capable of delaying the onset of the motor neuron disease and/or ameliorating negative effects of the motor neuron disease on motor function of the mammal to which it is administered.
 4. The method according to claim 1, wherein the oligopeptide is fused with a signal peptide for extracellular secretion and/or a tag peptide for purification and/or detection.
 5. The method according to claim 1, wherein the mammal is a human. 